Optimization of lipase production by Bacillus cereus ...

03

PLANT SCIENCES FEED ORIGINAL ARTICLE

2013

ISSN : 2231 - 1971 http://psf.lifescifeed.com

OPTIMIZATION OF LIPASE PRODUCTION BY BACILLUS CEREUS STRAIN MSU AS THROUGH SUBMERGED FERMENTATION.

ANANTHI S, IMMANUEL G AND PALAVESAM A Production of extracellular lipase in submerged culture of Bacillus cereus strain MSU AS has been investigated. The lipase production was optimized in shake flask experiments. The observed pH and temperature range optimum for maximum lipase production were 3–12 and 10–70°C, respectively. With a selected carbon sources, xylose (418.86 ± 0.4491) U/mL) and nitrogen source, peptone (335.43 ± 0.02041) U/mL) was suitable substrates for accelerating lipase production. The optimized concentration of xylose and peptone was 4g/100ml (389.25 ±0.1225) U/mL) and 2g/100ml (320.95 ± 0.2858) U/mL), respectively. Investigating the effect of metal ions and lipids showed that the MgSO4 and olive oil ((181.57 ± 0.0572) and (459.54 ± 0.0980) U/mL) was suitable substrates for maximizing lipase production, and the optimum concentrations registered were 0.12g/ 100 ml ((88.63 ± 0.1061) U/mL) and 1.2mL/100 ml (565.25 ± 0.0245) U/mL), respectively. KEYWORDS: Lipase, xylose, peptone, olive oil, Bacillus cereus strain MSU AS.

Cite this article as: Ananthi S, Immanuel G and Palavesam A. Optimization of lipase production by Bacillus cereus strain MSU AS through submerged fermentation. Plant Sciences Feed, 2013; 3 (2): 31-39 AUTHOR AFFILIATIONS: EMAIL :

Centre for marine science and technology, M. S. University, Rajakkamangalam (Tamil Nadu) India. 629502.

[email protected]

1. INTRODUCTION

2. MATERIALS AND METHODOLOGY

Triacylglycerol hydrolases (E.C. 3.1.1.3), also known by their trivial name as lipases, are enzymes that catalyse the hydrolysis of long-chain acylglycerols in aqueous emulsions [1]. Likewise, almost evidenced that, the lipase are also capable of catalyzing many reactions namely transesterification, interesterification and esterification between a fatty acid and an alcohol which is the reverse reaction of hydrolysis [2]. Lipases are used as additives in detergent formulations, in cleaning solutions and in waste treatment cocktails for downstream industrial processes and for domestic use also [3]. Similarly, reported that, the lipase producing microorganisms isolated from variety sources have been studied on the degradable efficiency as both single culture and mixed culture formula [4]. Lipases have been isolated from microorganisms especially from fungi, bacteria, and yeasts [5]. Similarly, many authors reported that, the common lipase-producing bacterial strains are Pseudomonas aeruginosa, P. fluorescens, Bacillus coagulans, B. cereus, Staphylococcus aureus, S. hyicus, Burkholderia glumae, B. cepacia, etc [6-8] productive fungal species belong to the genera Geotrichum, Penicillium, Aspergillus, Rhizopus, and Ophiostoma [9-10[. The yeasts include Yarrowia lipolytica, Candida rugosam, C. valida, Saccharomyces lipolytica, S. crataegenesis, Rhodotorula glutinis, Pichia bispora, and P. sivicola [11]. Along with evidenced that, the lipase units are generally measured based on their hydrolytic activity on tributyrin or triolein. However, the enzymes having a broad substrate specificity and the diversity of reactions catalyzed and make it difficult to define a universal test for lipase activity [12]. Many authors reported to, the bacterial and fungal strains were identified as a strong lipase producer, when streaked on Tween 80 agar medium, phenol red medium, Rhodamine B and spirit blue agar medium. These mediums are individually supplemented with Tween 80, olive oil and glycerol-tributyrin [13 – 15].

The bacterium used in this study was isolated from the gut of a marine fish Sardinella longicepsis collected from Colachal coast of Kanniyakumari District, Tamil Nadu, India. The bacterium produced a clear zone when it was streaked on Rhodanime B and Tween 80 agar plates supplemented with glycerol tributyrin and olive oil. The candidate bacterial strain was identified as Bacillus cereus MSU AS based on the standard key's of Bergey's manual of determinative bacteriology and molecular characterization (16S rRNA). The 16S rRNA sequence of Bacillus cereus MSU AS was compared with other similar bacterial groups by NCBI- BLAST data base program and then it was deposited in NCBI data bank (Accession no: JF-907013) [17[.

Plant Sciences Feed Vol. 3 Issue 2

Media used for lipase production consisted of Glucose 1.0g, yeast extract - 0.2g, peptone – 0.50g, KH2PO4 - 0.10g, MgSO4 – 0.02g, NaCl - 1.0g, olive oil -0.5 ml and pH - 7 (100ml). Cultivation was carried out in 250 ml Erlenmeyer flasks. The medium (100 ml) was inoculated with 4% inoculums. Fermentation was carried for 48h in an orbital shaker (150 rpm) at 37ºC. Sample were collected at 48h and centrifuged at 10,000 rpm for 15 min. the filtrated was considered as crude enzymatic extract and was used for enzyme assay. The similar culture conditions were followed for all the optimization experiments, and were carried out in triplicate. During the optimization, the experiments were performed in a consecutive manner by incorporating previously optimized parameters. The final optimization experiment was also performed under the previously optimized conditions. The interrelating effect of all optimized factors in this experiment was assessed. Lipase assay

Modification of lipase assay system consists of following ingredients 1ml of Tris HCl buffer (pH 7.2), 1ml of P-nitro phenyl palmitate (10mM) and 0.5 ml of culture supernatants [18]. Appropriate controls were also maintained. The mixture was incubated for 15min on a rotary shaker at 150 rpm at 37°C. Then the absorbance was taken at 400nm using UV-Vis Spectrophotometer (Techomb -8500) the amount of lipase production was found with the help of P-nitro phenol standard graph. One unit of lipase activity is equivalent to one microgram of Pnitro phenol released under standard assay condition. The optimization of medium components with suitable nutrient sources was carried out at the optimum pH and temperature, by substituting components present in the basal medium and subsequent optimization.

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Optimization of medium components for lipase production by Bacillus cereus strain MSU AS

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The effect of different growth media supplemented with various nitrogen and carbon sources including different dry products on lipase production by Candida rugosa was studied by Brockerhoff and Jensen [16]. Improvement of lipase production still depends on the optimization of culture conditions, including the composition of the culture medium such as carbon and nitrogen sources and on fermentation parameters such as dissolved oxygen, temperature and aeration rate. The primary objective of the present study was to screen microbes for the production of lipase in significant levels. This includes: (a) description of the techniques used to isolate and to screening lipase producing organism from gut of marine fish Sardinella longicepsis. (b) To determined the lipase production media by submerged fermentation.

Production of lipase in liquid media by Bacillus cereus strain MSU AS

Effect of different pH on lipase production by Bacillus cereus strain MSU AS To determine the optimum pH for lipase production, the pH of the production medium was varied viz 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 individually using 1N HCl and 1N NaOH. Then 2 ml of seed culture was inoculated in 50ml of sterile basal medium and incubated at 37ºC for 48h. After incubation the lipase production was assayed spectrophotometrically at 400nm.

Effect of different temperature on lipase production by Bacillus cereus strain MSU AS To determine the optimum temperature on lipase production, the different temperature such as 10, 20, 30, 37, 40, 50, 60 and 70ºC were selected. In the sterilized basal medium was inoculated with 2 ml seed culture and incubated individually in the respective temperature for 48h. After 48h of incubation, the lipase production in each flask was estimated using spectrophotometer (400nm). Effect of carbon sources on lipase production by Bacillus cereus strain MSU AS

The effect of carbon sources on lipase production was tested by using eleven different carbon sources, namely glucose, sucrose, fructose, maltose, cellulobiose, xylose, sorbitol, mannitol, starch gum and soluble starch. They were added individually in the basal medium at the concentration of 0.5g. Then the mediums were sterilized at 121ºC for 15 min and allow cool at 45ºC Finally the lipase production was determined by using Spectrophotometric assay method. The concentration of best carbon sources were investigated to determine the optimum concentration for maximum lipase production were 0.50, 1.0g, 1.5, 1.50, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0g /100 ml by Bacillus cereus strain MSU AS.

optimum concentration for maximum lipase production by Bacillus cereus strain MSU AS.

Effect of different metal ions on lipase production by Bacillus cereus strain MSU AS To select suitable metal ion (trace element) for lipase production by this bacterial strain, 0.02g of metal ions such as zinc chloride, magnesium chloride, barium chloride, mercuric chloride, cobaltus chloride, magnesium sulphate, Ethylene Diamine Tetra Acetic acid (EDTA), zinc sulphate, copper sulphate and ferric chloride were individually added into basal medium and inoculated with 2ml of the seed culture (Bacillus cereus strain MSU AS). The lipase production was measured at after 48h of incubation at 37°C in shake flask culture method. Special concentrations of best metal ions i.e. 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14 and 0.16g were calculated to obtain the highest lipase production by using Bacillus cereus strain MSU AS. Effect of triglycerides on lipase production by Bacillus cereus strain MSU AS

The lipase production was accelerated by incorporation of different lipid sources, namely palm oil, gingilly oil, olive oil, sunflower oil, neem oil, cod liver oil, coconut oil and castor oil in the optimized medium. They were tested individually at the fraction of 5ml (500µl) and the medium without oil was used as the control. In this experiment the assay was done regularly at intervals of 48h. In the respective time intervals, lipase production was determined by assay method. The volume of good triglycerides for Bacillus cereus strain MSU AS was investigated to determine the optimum volume for maximum lipase production were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 ml. 3. RESULTS

Effect of nitrogen sources on lipase production by Bacillus cereus strain MSU AS

In the present study, lipase producing bacterial strain was isolated from the gut of marine fish S. longiceps and then it was produce orange coloured fluorescent halos

33

(A) (B) Fig.1. Lipolytic activity of Bacillus cereus strain MSU AS in Rhodamine B agar plates (A) – Orange colour zone, (B) Negative for orange colour zone under UV light Screening of lipase production by using Rhodamine B and Tween 80 agar plate

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To test the effect of different nitrogen sources on lipase production by Bacillus cereus strain MSU AS, thirteen different organic and inorganic nitrogen sources were screened. They were yeast extract, soya meal, beef extract, skim milk powder, Tryptone, gelatin, peptone, sodium nitrate, ammonium chloride, ammonium nitrate, ammonium hydrogen carbonate, and potassium nitrate and ammonium sulphate. They were supplied individually in the basal medium at the concentration of 0.25g. Then the medium were sterilized and inoculated with 2 ml of 24h seed culture of Bacillus cereus strain MSU AS. The basal mediums were allowing incubation process for 48h at 37ºC. After 48h the fermentation basal mediums were used for determine the lipase production by using Spectrophotometric assay method. Different concentration of good nitrogen sources i.e. 0.25, 0.50, 0.75, 1.0, 1.25, 1.50, 1.75 and 2.0g /100 ml were studied to determine the


around the colony at 37ºC under the agar plate was expose to UV light at 350nm (Fig.1). And produced white clear crystal on the colonies, seen in Fig. 2, which shows positive results for lipase activity by using Tween 80 agar plate.

from Bacillus cereus strain MSU AS (Fig. 4). The one way ANOVA for the data on lipase production as a function of variation due to different pH is statistically highly significant (F = 1377585; P< 0.0001). 400

Lipase production (U/mL)

350 300 250 200 150 100 50 0 3

Identification of lipase positive strain by 16S rRNA sequencing Result on the present work of the BLAST research on the 16S rRNA sequence of the candidate strain showed 97% similarity to Bacillus cereus strain L12 (HQ398861). Then the sequence of the candidate bacterial strain Bacillus cereus MSU AS was submitted to GenBank in NCBI data base under the accession no JF 907013 (Fig. 3).

5

6

7

8

9

10

11

12

Different pH

Fig. 4.Effect of different pH on lipase production by Bacillus cereus strain MSU AS Effect of temperature on lipase production by Bacillus cereus strain MSU AS Temperature may affect lipase production [19]. In the present study, the highest lipase production (140.66 ± 0.0572) U/mL was noticed at 50°C by Bacillus cereus strain MSU AS (Fig. 5). The one way ANOVA for the data on lipase production as a function of variation due to different temperature is statistically more significant (F = 122523; P< 0.0001). 160 140 Lipase production (U/mL)

(A) (B) Fig. 2. Lipolytic activity of Bacillus cereus strain MSU AS in Tween 80 agar plates (A) - White colour precipitate formation, (B) - Negative for white colour precipitate formation

4

120 100 80

\

60 40 20 0 10

20

30

37

40

50

60

70

Different temperature (ºC)

Fig. 5. Effect of different temperature on lipase production by Bacillus cereus strain MSU AS

In the present study, the maximum lipase (364.58 ± 0.0572) U/mL was obtained at pH 8 up to 48h of incubation


34

Effect of pH on lipase production by Bacillus cereus strain MSU AS

The effects of different carbon sources, mainly carbohydrates and lipids, to support growth and lipase production by Penicillium restrictum in SSF. In the present study, the effect of various supplementary carbon sources on lipase production by Bacillus cereus strain MSU AS revealed that the xylose was influenced more than that of other tested carbon sources and it yielded 418.86 ± 0.449 U/mL lipase (Table. 1). The one way ANOVA for the data on lipase production as a function of variation due to different

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Fig. 3. Identification of Bacillus cereus strain MSU AS by using 16S rRNA through phylogenetic contraction ◊- Candidate bacterial strain Production of lipase in liquid media by Bacillus cereus strain MSU AS

Effect of different carbon sources on lipase production by Bacillus cereus strain MSU AS

350 300 250 200 150 100 50 0 0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Differenct concentrations of xylose (g/50mL)

Fig. 6. Effect of different concentrations of xylose on lipase production by Bacillus cereus strain MSU As Effect of different nitrogen sources on lipase production by Bacillus cereus strain MSU AS Like carbon sources, nitrogen source is another one important factor that would affect the lipase production by Bacillus cereus strain MSU AS. In the present study the effect of different nitrogen sources on lipase production by Bacillus cereus strain MSU AS revealed that highest lipase production (335.43 ± 0.2041) U/mL) was observed in nitrogen source peptone supplemented medium (Table. 1). The one way ANOVA for the data on lipase production as a function of variation due to different nitrogen sources is statistically highly significant (F = 964206.9; P< 0.0001). The effect of different concentrations of peptone on lipase production showed a positive linear increase (291.71 ± 0.0980) to (320.95 ± 0.2858) U/mL) with respect to the increase in the concentration of peptone from 0.50 to 1.0 g/100 ml at 0.5 g interval. Further increase in the concentration of peptone to 4g/ 100mL resulted in decreased lipase production (133.38 ± 0.057) U/mL) (Fig. 7). Lipase production (U/mL)

350 300 250 200 150 100

50 0 0.25

0.5

0.75

1

1.25

1.5

1.75

2

Different concentrations of peptone (g/50mL)

Fig.7. Effect of different concentrations of peptone on lipase production by Bacillus cereus strain MSU AS

Among the result of the present study also, maximum (181.57 ± 0.0572) U/mL lipase production was observed in MgSO4 supplemented medium by Bacillus cereus strain MSU AS through submerged fermentation (Table. 2). The one way ANOVA for the data on lipase production as a function of variation due to different metal ions is statistically more significant (F = 2471095; P< 0.0001). In the same way, the different concentrations of MgSO4 (0.0, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14 and 0.16g) were tested on lipase production (Fig. 8). Among the tested concentrations, maximum (188.63 ± 0.1061) U/mL) lipase production was recorded at 0.06g/50mL concentration. On the other hand, minimum lipase production was obtained at 0.16g (137.48 ± 0.0653) U/mL) respectively. The one way ANOVA for the data on lipase production as a function of variation due to different concentration of MgSO4 is statistically very significant (F = 126860.5; P< 0.0001). 250 200 150 100 50 0 0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Different concentrations of MgSO4 (g/50mL)

Fig. 8. Effect of different concentrations of MgSO4 on lipase production by Bacillus cereus strain MSU AS Effect of different triglycerides on lipase production by Bacillus cereus strain MSU AS Results indicated that among the tested triglycerides, olive oil was supported for maximum lipase (459.54 ± 0.0980) U/mL production by Bacillus cereus strain MSU AS. Since, 0.1ml of olive oil contributed maximum lipase production; further the effect of different volume of olive oil was tested for lipase production. It resulted that 0.6ml (1.2%) olive oil was found to influence maximum (565.25 ± 0.0245) U/mL lipase production by Bacillus cereus strain MSU AS (Table. 2). The one way ANOVA for the data on lipase production as a function of variation due to different triglycerides is statistically very significant (F = 3722405; P< 0.0001). Similarly, the effect of various volumes of olive oil ranged between 0.1 – 1ml on lipase productions (Fig. 9).

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400

Effect of different metal ion on lipase production by Bacillus cereus strain MSU AS

Page

450

The one way ANOVA for the data on lipase production as a function of variation due to different concentration of peptone is statistically very significant (F = 476307.8; P< 0.0001).


carbon sources is statistically very significant (F =534426.4; P< 0.0001). On the same way, in the present study was optimization of lipase production by Bacillus cereus strain MSU AS giving positive effect when the culture medium was supplied with 2.0g (Xylose) (Fig. 6). The one way ANOVA for the data on lipase production as a function of variation due to different concentration of xylose is statistically more significant (F = 168452.6; P< 0.0001).


Fig. 9. Effect of different volume of olive oil on lipase production by Bacillus cereus strain MSU AS

Lipase production (U/mL

700 600 500 400 300 200 100 0 0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Different volume of olive oil (mL/50mL)

After 48 h of incubation, maximum lipase production of 565.25 ± 0.0245 U/mL was recorded in the medium containing 0.6 ml/50ml of olive oil by Bacillus cereus strain MSU AS. The one way ANOVA for the data on lipase production as a function of variation due to different volume of olive oil is statistically highly significant (F = 3864069; P< 0.0001).

Table. 1. Effect of different carbon and nitrogen sources on lipase production by Bacillus cereus strain MSU AS Carbon sources (0.5g/50mL) Glucose Sucrose Fructose Maltose Cellulose Xylose Sorbitol Mannitol Starch gum Soluble starch Lactose Control -

Lipase production (U/mL) 127.66 ± 0.1225 246.46 ± 0.2041 173.50 ± 0.0980

Nitrogen sources (0.25g/50 mL) Yeast extract Soya meal

Beef extract

152.33 ± 0.0735

Skim milk powder

418.86 ± 0.4491

Gelatin

205.55 ± 0.0653

Tryptone

230.78 ± 0.1388

Peptone

220.36 ± 0.1796

NH4Cl2

227.32 ± 0.0980 124.52 ± 0.0816 -

NH4SO4

211.00 ± 0.0898

-

-

328.56 ± 0.2041 306.05 ± 0.2286 300.93 ± 0.2205 331.65 ± 0.2286 324.18 ± 0.1225 48.196 ± 0.0098 132.41 ± 0.2286 142.78 ± 0.1470

NH4NO3

NH4HCO3

264.40 ± 0.0980

335.43 ± 0.2041

NaNO3

57.235 ± 0.0098


262.23 ± 0.1225

KNO3

131.90 ± 0.0898

Control

120.09 ± 0.1470

12.406 ± 0.0098

Table. 2. Effect of different metal ions and triglycerides on lipase production by Bacillus cereus strain MSU AS

BaCl2 BaSO4 COCl2 MgSO4 ZnSO4 EDTA CuSO4 FeCl3 Control

Triglycerides (0.5mL/50mL) Olive oil

156.67 ± 0.0898

Palm oil

141.77 ± 0.0735

Sun flower oil

174.47 ± 0.0163

Gingilly oil

181.57 ± 0.0572

Cod liver oil

137.85 ± 0.0653

Castor oil

16.695 ± 0.0098 96.023 ± 0.0833 36.665 ± 0.0098 37.911 ± 0.0098 25.86 ± 0.0980

4. DISCUSSION Screening of lipase production by using Rhodamine B and Tween 80 agar plate Most commercial lipases are produced from microbes [20]. In the present study, lipase producing bacterial strain was isolated from the gut of marine fish S.


Lipase production (U/mL) 459.54 ± 0.0980 44.460 ± 0.0147 77.851 ± 0.0988 99.482 ± 0.1804 204.91± 0.0653

Neem oil

122.58 ± 0.1470

Coconut oil

4.2892 ± 0.0105

Control

162.85 ± 0.1225 18.817 ± 0.0147

-

-

longiceps and then it was produce orange coloured fluorescent halos around the colony at 37ºC under the agar plate was expose to UV light at 350nm. And produced white clear crystal on the colonies. which shows positive results for lipase activity by using Tween 80 agar plate. Likewise, evidenced that, the production of lipase activity by Pseudomonas strain was verified on both LB (Luria

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MgCl2

Lipase production (U/mL) 34.313 ± 0.0098

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Metal ions (0.02g/50mL) ZnCl2

In the present study, BLAST research on the 16S rRNA sequence of the candidate strain showed 97% similarity to Bacillus cereus strain L12 (HQ398861). Then the sequence of the candidate bacterial strain Bacillus cereus MSU AS was submitted to GenBank in NCBI data base under the accession no JF 907013. Similarly, isolated alkaline thermostable lipase producing bacterial strain from spoiled coconut and identified it based on 16S rRNA sequencing as Bacillus cereus (AB 24464) which had nearest homology with Bacillus thuringiensis (EF 210288) in NCBI BLAST search [22]. Production of lipase in liquid media by Bacillus cereus strain MSU AS

Effect of pH on lipase production by Bacillus cereus strain MSU AS In the present study, the maximum lipase (364.58 ± 0.0572) U/mL was obtained at pH 8 up to 48h of incubation from Bacillus cereus strain MSU AS. In the same way, the maximum lipase production (13.75) EUm-1 was occurring in the lipase production medium at pH 8.0 by Acinetobacter baylyi through response surface methodology (RSM) [23]. Likewise, the high lipase production (4.41) U/mL was obtained from Acinetobacter sp. RAG-1 in a production medium at pH 8.0 through submerged fermentation [24]. Effect of temperature on lipase production by Bacillus cereus strain MSU AS

In the present study, the highest lipase production (140.66 ± 0.0572) U/mL was noticed at 50°C by Bacillus cereus strain MSU AS. In the same way, the highest amount of lipase production (49.5 ± 1.7) U/mL was found to be at 50°C by Bacillus sp. through submerged fermentation [25]. Similarly, the maximum lipase production (12.8) EUm-1 was observed at 50°C by Acinetobacter baylyi through response surface methodology (RSM) [23]. Effect of different carbon sources on lipase production by Bacillus cereus strain MSU AS

In the present study, the effect of various supplementary carbon sources on lipase production by Bacillus cereus strain MSU AS revealed that the xylose was influenced more than that of other tested carbon sources and it yielded 418.86 ± 0.449 U/mL lipase. Likewise, the maximum lipase was (4.7 ± 0.14 U (ml medium)-1) obtained in 1% of xylose supplemented medium as good carbon sources for lipase production by Rhizopus

Effect of different nitrogen sources on lipase production by Bacillus cereus strain MSU AS

In the present study the effect of different nitrogen sources on lipase production by Bacillus cereus strain MSU AS revealed that highest lipase production (335.43 ± 0.2041) U/mL) was observed in nitrogen source peptone supplemented medium. In the same way, the maximum lipase production (810) units/mL) was observed in peptone supplemented medium by Bacillus megaterium AKG -1 through submerged fermentation [28]. The effect of different concentrations of peptone on lipase production showed a positive linear increase (291.71 ± 0.0980) to (320.95 ± 0.2858) U/mL) with respect to the increase in the concentration of peptone from 0.50 to 1.0 g/100 ml at 0.5 g interval. Further increase in the concentration of peptone to 4g/ 100mL resulted in decreased lipase production (133.38 ± 0.057) U/mL). Similarly, the strain of Pseudomonas aeruginosa KM110 was produced maximum (0.17 U/mL) lipase by using 2g of peptone supplemented medium through submerged fermentation [29].

Effect of different metal ion on lipase production by Bacillus cereus strain MSU AS In the present study also, maximum (181.57 ± 0.0572) U/mL lipase production was observed in MgSO4 supplemented medium by Bacillus cereus strain MSU AS through submerged fermentation. Similarly, the maximum lipase production was observed in medium supplemented with 0.16g MgSO4 by Bacillus coagulans [30]. In the same way, the different concentrations of MgSO4 (0.0, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14 and 0.16g) were tested on lipase production (Fig. 8). Among the tested concentrations, maximum (188.63 ± 0.1061) U/mL) lipase production was recorded at 0.06g/50mL concentration.

Effect of different triglycerides on lipase production by Bacillus cereus strain MSU AS Results indicated that, the present study olive oil was supported for maximum lipase (459.54 ± 0.0980) U/mL production by Bacillus cereus strain MSU AS. Since, 0.1ml of olive oil contributed maximum lipase production; further the effect of different volume of olive oil was tested for lipase production. It resulted that 0.6ml (1.2%) olive oil was found to influence maximum (565.25 ± 0.0245) U/mL lipase production by Bacillus cereus strain MSU AS. In agreement with the present finding, the maximum (4.25) EU/mL amount of lipase was obtained in 1.5% of olive oil supplemented medium at 37ºC for 48h incubation by

37

Identification of lipase positive strain by 16S rRNA sequencing

oligosporus through submaerged fermentation [26]. On the same way, in the present study was optimization of lipase production by Bacillus cereus strain MSU AS giving positive effect when the culture medium was supplied with 2.0g. Similarly, the maximum lipase production (413.84) U/mL) was observed in 1% of xylose supplemented medium under solid state fermentation by Aspergillus niger [27].

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Bertani) and MM (Minimal medium) agar containing high refined olive oil and fluorescent dye Rhodamine B at both 25ºC and 4ºC. Orange colour fluorescent halos around lipase producing colonies were seen when theses agar plated were expose to UV light at 350nm [21].


5. CONCLUSION

Lipases have many industrial applications, in the present study, an attempt was made to isolate and identify a novel lipase producing bacterium Bacillus cereus strain MSU AS from the gut of a marine fish S. longicepsis. The lipase production by the candidate bacterial strain through SmF was found to be accelerated at optimized culture conditions such as pH, temperature and various substrates concentrations. From the result it could be concluded that, the medium temperature of 50ºC and pH of 8 were optimum for maximum lipase production. The optimum substrates required for enhancing production of lipase were 4% xylose, 2% peptone, 1.2% olive oil and 0.12% MgSo4. Then determine the 49KDa molecular weight of purified lipase protein and lipase activity (Zone formation). Through this study, it could be confirmed that Bacillus cereus strain MSU AS is a potential strain for lipase production through SmF.

6. REFERENCES [1] Lee HK, Min JA, Sung HK, Won HS, Byeong CJ (2003). Purification and characterization of cold active lipase from psychrotrophic Aeromonas sp. LPB4. J Microbiol. 41:22–7. [2] Macraea, R. (1983). Extracellular microbial lipases. In Microbial Enzymes and Biotechnology, pp. 225-250. Edited by W. M. Fogarty. London : Applied Science Publishers. [3] Lai O.M., Akoh C.C., Weete J.D. (2007). Microbial Lipases. In C.C. Akoh and D. B. Min (eds.) Food Lipids, 4th edn. Taylor &Francis Group, USA. [4] Bhumibhamon, O., Koprasertsak, A and Funthong, S (2002). Biotreatment of High Fat and Oil Wastewater by Lipase Producing Microorganisms. Kasetsart J. (Nat. Sci.) 36 : 261 – 267. [5] Saeed AEM, Ahmed BM, Abdelkarim EI, Ibrahim KEE, Mohamed, OSA (2005). New anti cestodal drugs : Efficacy , toxicity and structure activity relationship of some derivatives of the open lactam form of praziquantel ( N – sulphanilamido / COO – alkyl). J. Sci.Technol. 6: 109-119. [6] Simons JW, van Kampen MD, Riel S, Gotz F, Egmond MR, Verhey HM (1998). Cloning, purification and characterization of the lipase from Staphylococcus epidermidis—comparison of the substrate selectivity with those of other microbial lipases. Eur J Biochem . 253:675–83. [7] Kim HK, Park SY, Lee JK, Oh TK (1998). Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1. Biosci Biotechnol Biochem. 62: 66–71. [8] Ito, T., H. Kikuta, E. Nagamori, H. Honda, H. Ogino, H. Ishikawa. and T. Kobayashi. (2001). Lipase production in two-step fed-batch culture of organic solventtolerant Pseudomonas aeruginosa LST-03. J Biosci Bioeng 91: 245- 50.


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